The present invention relates to liquid chromatography, and more specifically to an improved system for controlling the rate of change characteristic, or gradient, between two eluents flowing into a liquid chromatography column.
Chromatography, broadly stated, is an analytical procedure which comprises a method for separating constitutents in a mixture, wherein it is desired to identify certain or all of the constituents. Basically, the identity of the constituents is determined by monitoring the relative transit time of the various constituents through a separation element, or column. The column is packed with a material such as a treated resin, and the sample is transported through the column by means of a mobile phase or carrier, termed an eluent. As the eluent sweeps the sample through the column, various constitutents of the sample are selectively adsorbed and desorbed by material of the column. The rate of adsorption and succeeding release of the adsorbed component into the flowing eluent, depends upon several parameters including the nature of the column material, and of the component itself. Accordingly, certain components are adsorbed and desorbed more rapidly than others, and accordingly exit at the distal end of the column at an earlier time. A sensor at the distal end of the column is responsive to the appearance of separated constituents and produces output signals indicating their passage. The transducer, however, is usually itself incapable of distinguishing among the constituents; their identity is inferred from their relative time of arrival.
It will be appreciated that in order to infer the identity of the constituents, their arrival times must not occur all at once. The "peaks" which correspond to transducer signals indicating the passage of separated constituents must be discrete in time so that each of the various constituents may be identified.
Due to the irregular nature of the adsorption/desorption characteristics of the constituents of any given sample, it often occurs that some constituents pass through the chromatograph column at approximately the same rate. Several techniques have been devised in order to separate the closely-placed peaks, without unduly expanding the length of the sample run time. Indeed, if the process is unduly lengthened the resolution or "sharpness" of the detected peaks may degenerate to such an extent that the presence of a peak is difficult to ascertain. Aside from this it is not desirable to occupy analytical equipment for lengthy periods and accordingly it is desirable to minimize run times.
One of the techniques which has been developed for separating constituent peaks in liquid chromatography is termed gradient elution. With such a process a plurality, preferably two, eluents are used to carry a sample through the sample column. The proportion of the eluents is changed as a function of time starting, for instance, with a 100% concentration of a first eluent and changing the proportion until, near the end of the run, the second eluent constitutes 100% of the eluent being used. The rate of change of the ratio between the two eluents is termed the gradient; a constant rate of change results in a linear gradient, while a variable rate of change may produce non-linear, e.g. exponential, gradients. It has been found that in many instances exponential gradients are extremely useful since, for example, an initial part of a sample run may take place with a first eluent ratio which varies only slowly; while the latter portion of the run can occur under conditions wherein the eluent ratio varies rapidly. Such changes in the eluent gradient have been found to be highly advantageous in producing the desired peak separations.
In order to provide the desired non-linear gradient, apparatus have been derived for automatically varying the proportion of two eluents. For example, in U.S. Pat. No. 3,446,057-Bakalyar et al a system is shown wherein a pair of motor-driven pumps are operated by a voltage supplied from a function generator. Systems such as that disclosed in the Bakalyar patent, while serving to advance the state of the art have been found to provide only fixed-length gradients whose overall characteristic has not only a predetermined shape, but a fixed time constant. If it is desired, for example, to provide an exponential eluent gradient which extends between two fixed non-terminal eluent ratios the exponential gradient is conventionally "switched in" at a desired initial point and then "switched out" at a termination point. This truncates the curve so that it starts and ends abruptly at "plateaus". It has been found that the latter approach substantially negates the value of the exponential curve, and moreover makes it impossible to duplicate the overall curve characteristic between differing initial and termination ratios or curve end points.
After a run is terminated the chromatograph system is conventionally reset by switching back to the original eluent which is used at the start of a run. In the example given above, this is done by switching from a 100% concentration of the first, initially-used eluent. This is usually accomplished by the simple expedient of opening or closing a valve so that the first eluent alone is allowed to flow. Recently it has been found that a sudden, abrupt "resetting" from one eluent to another is detrimental to at least some liquid chromatograph columns. It will accordingly be understood that it would be highly desirable to provide an elution gradient control system for effecting a curve of a desired configuration between varying initial and final eluent ratios; and for controlling the resetting time therebetween.
It is therefore an object of the present invention to provide an eluent control system for effecting a uniform gradient configuration between varying limits.
It is another object of the invention to provide an apparatus for achieving various gradient configurations over controllable time periods.
Another object is to provide a method for producing a consistent, repeatable eluent gradient which may be expanded or contracted without changing the overall shape thereof.
Still another object of the invention is to provide a control means for controllably resetting an eluent ratio from a final to an original value without damaging a chromatography column.
Still another object is to provide a method for returning an eluent composition from a final to an initial value along a predetermined exponential curve.
It is yet another object of the present invention to vary eluent composition along an exponential curve in a first time limit, and subsequently return the eluent composition to its initial state according to a similar controlled gradient, in a second, shorter period of time.